A number of analytical procedures have been developed in the biochemical art wherein it is required to concentrate and remove low molecular weight ions or solutes from peptide solutions in order to have a relatively pure concentrated peptide sample which can be analyzed effectively. Many other analytical procedures, involving not only peptides but macromolecular species in general, also have been developed wherein it is necessary to concentrate and/or “desalt” a macromolecular component in a liquid sample, as there is commonly a need in biochemistry/medicinal chemistry for relatively pure analytes devoid of salts, detergents and other contaminants. The presence of contaminating substances can be deleterious, in that they often interfere with subsequent chemical or physical analyses. Analogous situations exist in the environmental art and in classical chemical analysis.
Ultrafiltration devices are commonly used for the “purification” and/or sample preparation of biomolecules and natural products. For such a process to be successful, a membrane must be selected that retains the molecules of interest, yet passes the impurities. Although this scenario is relatively straightforward for analytes greater than about 10,000 molecular weight, it becomes increasingly problematic for substances less than about 5000 molecular weight. The reason is due to the fact that the required membrane porosity to retain the 5000 molecular weight analyte is so low that the water permeability (flow rate) becomes poor and processing times too long. For example, a typical centrifugal “spin time” for a device using a membrane suitable for analytes having a molecular weight of 30,000 or more is about one hour, whereas as many as six hours may be required for analytes of about 1000 molecular weight using a suitable membrane. Furthermore, such long-term exposure to high g-forces frequently results in device failure.
Sample processing with vacuum as the driving force offers advantages over centrifugation. Collection of the elution volume by centrifugation of multiwell formats is possible but difficult, since the volume in each well may vary due to rapid evaporation during transfer of the multiwell plate to the centrifuge and especially during centrifugation. Moreover, every time the protein sample is transferred, such as from pipette to collection plate, or is resuspended, sample is lost to due adherence to the interfaces of these devices. Since sample amounts are typically in the femtomole range, sample losses are unacceptable. Furthermore, centrifugation is also not amenable to automation, as the plate must be manually placed and removed into and from the centrifuge.
The sample quantities of protein now common in the art are in the 0.01 to 10 microgram range and smaller. At such low loads, efficient sample handling is crucial to avoid loss. Conventional methods and devices based on ultrafiltration for sample preparation are not practical for handling the “microseparation” of such small sample volumes. However, the application of adsorption technology at this scale could offer a highly effective approach to micro-mass sample preparation.
One conventional method for making sample preparation devices that contain chromatographic media is to first insert a precut porous plug obtained from, for example, a porous plastic frit, or a fiberous glass or cellulose sheet into a well or the tip of a pipette, followed by the addition of loose particles and a second porous plug. The plugs serve to retain the particles in place in the pipette tip or well. However, the plugs also entrap excess liquid thereby creating dead space or volume (i.e., space not occupied by media or polymer that can lead to poor sample recovery, contamination such as by sample carry-over, etc.). The frits also provide an increased surface area that will increase the binding losses. Also, the frits increase the required elution volume thereby reducing the concentration of the eluant sample. This procedure cannot be used with extremely small liquid delivery devices such as pipette tips, as there is no practical way to load either the plug or the particles to obtain a micro-adsorptive device that contains 1 milligram or less of adsorbent to be used for the aforementioned extremely small sample loads.
Alternatively, a micro sample preparation device can be made by lodging media in the bore of a capillary pipette. However, the flow through such devices is typically slow and variable.
Moreover, although from a mass adsorption standpoint, adsorptive powders offer the highest capacity, they are difficult or indeed impossible to handle in milligram quantities. Although polymer-based adsorptive membrane sheets are relatively easy to handle, even this approach has difficulties at extremely small scales. Moreover, the binding capacity of the membrane is poor as a result of relatively low substructure surface area.
Current trends in research are to analyze larger and larger numbers of smaller samples using automated handling equipment or robotics. Quantities of individual samples are from the nano-mole levels to atto-mole levels. As a result, instrumentation is becoming more sensitive and a need exists for sample handling formats to be miniaturized, high density and disposable.
Sample preparation prior to analysis (such as by MALDI TOF mass spectrometry) often involves desalting and concentration of samples (e.g., peptides) down to a 1-2 microliter volume. These volumes are likely to decrease to nanoliter volumes in time. Simultaneous preparation and analysis of multiple samples is often desirable. Multiwell plates have been developed for simultaneous assay, typically consisting of 96, 384 or 1536 reaction vessels or wells per plate.
Certain sample preparation devices, such as the ZipTip® device commercially available from Millipore Corporation, are excellent tools for sample preparation prior to MALDI analysis. They are a single sample processor that can be used to spot sample onto the MALDI target manually or by automated equipment. More specifically, U.S. Pat. Nos. 6,048,457 and 6,200,474 (the disclosures of which are hereby incorporated by reference) teach the formation of cast membrane structures for sample preparation that are formed by phase inversion of a particle loaded polymer system at the housing orifice. The polymer is precipitated when the portion of the housing (containing the soluble polymer/particle lacquer) is immersed in a precipitation bath (typically water). The insertion creates a slight liquid pressure across the lacquer such that the water intrudes upon the polymer creating an open sponge-like structure upon precipitation. However, at the polymer-water interface on the structure there is a semi-permeable membrane film that creates a high resistance to flow. When this barrier is either abraded or cut off, the resulting structure is highly permeable. The resulting device is suitable to allow flow under the low differential pressures generated by a common 10 microliter hand-held pipettor (e.g. Gilson, Pipetman). However, the macroporous structure is less efficient at capturing solutes. As such, often times the sample volume must be passed through the membrane structure multiple times.
It would be desirable to provide a sample preparation device and method that includes a membrane structure that has a more dense structure, better suited for formats such as a vacuum plate, that must capture solutes in a single pass.
It also would be desirable to provide a process for preparing a high-throughput sample preparation device and the device so made.
It is therefore an object of the present invention to provide a sample preparation device that can concentrate, purify and/or desalt molecules from sample solutions.
It is another object of the present invention to provide a sample preparation device that can concentrate, purify and/or desalt molecules and elute in small sample solutions.
It is another object of the present invention to provide a sample preparation device that can concentrate, purify and/or desalt molecules from sample solutions in a variety of form geometries.
It is a still further object of the present invention to provide a sample preparation device that is simple and economic to manufacture.
It is yet a further object of the present invention to provide a method of directly forming membrane structures in a housing spout, wherein both the spout and the reservoir of the housing have narrow geometries.
It is a further object of the present invention to provide a high density membrane that assumes the shape of the housing or portion thereof in which it is cast, and can be retained in that housing by chemical adhesion without the use of porous plugs.
It is a still further object of the present invention to provide a multi-well device wherein various arrays within the array of wells contain a high density of composite membrane having the same or different chemistries.
It is yet a further object of the present invention to provide a single or multi-well device having a high density membrane, with or without adsorptive particles contained therein, that assumes the shape of the housing or a portion thereof in which it is cast and can be retained in that housing without the use of porous plugs.